Cyclic nucleotide phosphodiesterases (PDEs) comprise 5 major classes with distinctive physicochemical, immunological, structural and kinetic properties. One family, the so-called cGMP-inhibited low km cAMP PDE (cGI PDE) is characterized by its high affinity for cAMP and cGMP and specific inhibition of its cAMP hydrolysis by cGMP and inotropic/vasodilatory agents (milrinone, cilostamide, fenoximone...). The cGI PDEs play an important role in the regulation of myocardial contractility, platelet aggregation and the antilypolitic action of insulin. A cGI PDE purified from outdated human platelets using ion-exchange and affinity chromatography exhibited 2 major bands (approximately 63 kDa and 53-55 kDa doublet) on SDS PAGE or Western immunoblots. Rabbit polyclonal anti-platelet PDE antibody used in Western blot and immunoprecipitation studies cross-reacted with cGI PDE from human cardiac sarcoplasmic reticulum and rat adipocyte microsomes. Based on partial amino acid sequence of peptides from platelet cGI PDE oligonucleotide probes were synthesized and used to screen erythroleukemic cell and human heart cDNA libraries. Several positive cDNA clones from the human heart library were isolated and sequenced. One clone contains a approximately 4 kb fragment with an open reading frame encoding a protein of approximately 125 kDa. Another clone, encoding a approximately 54 kDa truncated fragment including most of the carboxyterminal region of the cGI PDE was expressed as a glutathione-S-transferase (GST)-fusion protein. The activity of the expressed truncated PDE was inhibited by cGMP and cilostamide. The sequences of these clones and Northern and Southern analysis will enhance our understanding of human platelet and human cardiac cGI PDE structure/function and regulation of gene expression and perhaps in design of specific and effective drug therapies.